1. TECHNICAL FIELD
The present invention relates to a novel hyaluronic acid gel and a method of its production, and further, to a biomedical material with good biocompatibility.
2. BACKGROUND ART
Hyaluronic acid is a linear macromolecular polysaccharide consisting of alternately bonded xcex2-D-N-acetylglucoamine and xcex2-D-glucuronic acid. Hyaluronic acid is found not only in connective tissues of mammals but also in cockscombs and the capsules of streptococci. Hyaluronic acid is obtainable not only by extraction from cockscombs and umbilical cords, but also as purified products from the culture broth of streptococci.
Natural hyaluronic acid is polydisperse in respect of molecular weight and is known to show excellent biocompatibility even when implanted or injected into the body by virtue of the absence of species and organ specificity. Further, because of the drawbacks of hyaluronic acid in biological application attributable to the easiness of dissolution in water such as the relatively short in vivo residence time, various chemical modifications of hyaluronic acid have been proposed.
A representative of them is a high-swelling crosslinked hyaluronic acid gel obtained by using a bifunctional crosslinker such as divinyl sulfone, a bisepoxide or formaldehyde (U.S. Pat. No. 4,582,865, JP-B-6-37575, JP-A-7-97401 and JP-A-60-130601).
A chemical modification of hyaluronic acid utilizing the solubility of tetrabutylammonium hyaluronate in organic solvents such as dimethyl sulfoxide has been disclosed, too (JP-A-3-105003). Formation of ester linkages between the carboxyl groups and the hydroxyl groups in hyaluronic acid by treating tetrabutylammonium hyaluronate with triethylamine and 2-chloro-1-methylpyridinium iodide in dimethyl sulfoxide has also been disclosed (EP-A-0341745A1).
Further, as an approach to insolubilization of hyaluronic acid in water without using covalently binding chemicals, preparation of a hyaluronic acid-polymer complex by ionically bonding hyaluronic acid and a polymer having an amino or imino group via the carboxyl groups in hyaluronic acid and the amino or imino group in the polymer has been disclosed (JP-A-6-73103).
It is known that a hyaluronic acid aqueous solution forms a so-called putty gel by jellying when acidified, for example, to pH 2.0-2.7, but no putty gel is formed at a pH below 2.
The putty gel is differentiated from the hyaluronic acid gel according to the present invention by its quick dissolution in a neutral aqueous solution.
As another approach, production of a hyaluronic acid gel from a hyaluronic acid aqueous solution in the presence of 20-80 wt % of a water-miscible organic solvent at pH 2.0-3.8 has been disclosed (JP-A-5-58881). However, it is also disclosed that the resulting hyaluronic acid gel dissolves in water with no coating on it.
Further, some general methods of producing polymer gels by repeatedly freezing and thawing aqueous solutions of polymers represented by polyvinyl alcohol and glucomannan have been proposed (JP-A-57-190072 and JP-A-5-161459).
Although freezing-thawing and freeze-drying are widely used as general techniques for purifying or preserving hyaluronic acid or biogenic samples containing hyaluronic acid, no report has been made on formation of a hyaluronic acid gel by such techniques yet because they are usually used under neutrality control.
Hyaluronic acid has extraordinarily high viscosity and good moisture retentivity, and is intrinsically devoid of antigenicity and highly biocompatible. Therefore, it is used as a therapeutic medicine for osteoarthritis and as a supplementary material in ophthalmic surgery.
Use of hyaluronic acid itself as a postoperative adhesion preventive has also been studied. However, hyaluronic acid does not have much effect due to the relatively short in vivo residence time and diffusively drains away from the wound surface in a short time due to its water solubility (Journal of Gynecologic Surgery vol. 7, No. 2, 97-101(1991)).
Modification of carboxymethyl cellulose and sodium hyaluronate with a carbodiimide crosslinker on the basis of JP-A-5-508161 and JP-A-6-508169 afforded the development of an adhesion preventive film xe2x80x9cSeprafilmxe2x80x9d (Genzyme).
Despite attempts to utilize the outstanding biocompatibility intrinsic to hyaluronic acid to the maximum, no hyaluronic acid gel usable as a biocompatible biomedical material with a long in vivo residence time has been developed yet without any chemical crosslinkers or chemical modifiers or formation of complexes with cationic polymers.
The present inventors have conducted extensive research on the physicochemical properties of hyaluronic acid itself and consequently have found that a hyaluronic acid gel can be obtained by freezing and thawing at least once a hyaluronic acid aqueous solution adjusted to a specific pH. They have also found that the hyaluronic acid gel thus obtained dissolves in water very slowly.
Conventional modifications of hyaluronic acid have an inevitable problem of extra risks such as toxicity and bioincompatibility intrinsic to the modifications because of the use of chemical reactants despite numberless efforts.
For example, chemical modification, crosslinking or ionic treatment of hyaluronic acid with a metal salt may afford adhesion preventives with improved in vivo persistency. However, the resulting adhesion preventives no longer retain the structure of natural hyaluronic acid and are not essentially the same as natural hyaluronic acid in respect of physiological effects, biocompatibility and safety inclusive of toxicity, because of the crosslinkers or metals covalently or ionically bound in the hyaluronic acid molecules. In addition, it has been difficult to completely circumvent the problems of the residual toxicity of these crosslinkers and the risk of decomposition products of crosslinkers to the body.
The present inventors have found that the hyaluronic acid gel according to the present invention has ideal biocompatibility and persistency as a biomedical material, particularly ideal biocompatibility and persistency as an adhesion preventive and markedly prevents postoperative adhesion. The present inventors have accomplished the present invention on the basis of this discovery.
The present invention provides (1) a gel made of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution, (2) the hyaluronic acid gel according to (1), which keeps its shape for at least one day in a neutral aqueous solution at 25xc2x0 C, (3) the hyaluronic acid gel according to (1), which dissolves in a neutral aqueous solution at 25xc2x0 C. in one day to a degree of dissolution of at most 50%, (4) the hyaluronic acid gel according to (1), which dissolves in a neutral aqueous solution at 37xc2x0 C. in 12 hours to a degree of dissolution of at most 50%, (5) the hyaluronic acid gel according to (1), which dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (6) the hyaluronic acid gel according to (1), which is formed by freezing and then thawing an aqueous solution of hyaluronic acid at pH 3.5 or below, (7) a method of producing the hyaluronic acid gel according to (6), which comprises adjusting an aqueous solution of hyaluronic acid to pH 3.5 or below, and freezing and thawing the solution at least once, (8) a biomedical material containing a gel made of hyaluronic acid alone which satisfies the following requirements (a) and (b): (a) the hyaluronic acid gel dissolves in a neutral aqueous solution at 25xc2x0 C. in one day to a degree of dissolution of at most 50%, and (b) the gel dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (9) the biomedical material according to (8), wherein the gel made of hyaluronic acid alone is sheet-like, filmy, flaky, spongy, massive, fibrous or tubular, (10) a biomedical material containing a hyaluronic acid gel and un-gelled hyaluronic acid, wherein the hyaluronic acid gel dissolves in a neutral aqueous solution at most 50%, and the hyaluronic acid gel dissolves to yield solubilized hyaluronic acid having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of hyaluronic acid, (11) a biomedical material containing a hyaluronic acid gel made of hyaluronic acid alone which is sheet-like, filmy, spongy, massive, fibrous or tubular and un-gelled hyaluronic acid, and (12) the biomedical material according to any one of (8) to (11), which is an adhesion preventive.